Currently, the choice of medical treatment for major depressive disorder (MDD) is primarily based on a trial-and-error process. Thus, identification of individual factors capable of predicting treatment response is of great clinical relevance. Recent work points towards beclin-1 and inflammatory factors as potential biomarkers of antidepressant treatment response. The primary aim of the study was to investigate whether pre-treatment serum levels of beclin-1 and inflammatory factors could predict antidepressant treatment response in Chinese Han patients with MDD. Forty patients with MDD were treated with either a selective serotonin reuptake inhibitor (SSRI) (paroxetine in 20 cases) or a serotonin–norepinephrine reuptake inhibitor (SNRI) (duloxetine in 13 cases and venlafaxine in 7 cases). Depression scores and serum levels of beclin-1 were measured at the baseline and after 8 weeks of antidepressant treatment. Serum C-reactive protein (CRP), interleukin (IL)-1B, and IL-6 levels were determined using enzyme-linked immunosorbent assay kits at the baseline. Twenty-seven patients were identified as treatment responders, whereas 13 were identified as non-responders after 8 weeks of antidepressant treatment. Baseline serum beclin-1 levels were significantly higher in non-responders than in responders (p = 0.001), whereas no differences were found in baseline serum CRP, IL-1B, or IL-6 levels between responders and non-responders. There were no significant correlations between baseline levels of beclin-1 and baseline IL-1β, IL-6, and CRP levels—neither in the total sample nor in responder and non-responder groups. Moreover, logistic regression models and a random forest model showed that baseline serum beclin-1, but not inflammatory factors, was an independent and the most important predictor for antidepressant treatment response. Furthermore, serum beclin-1 levels were significantly increased in responders (p = 0.027) but not in non-responders after 8 weeks of treatment (p = 0.221). Baseline serum beclin-1 levels may be a predictive biomarker of antidepressant response in patients with MDD. Moreover, beclin-1 may be involved in the therapeutic effect of antidepressant drugs. Major depressive disorder (MDD) is a severe and debilitating illness that is estimated by the World Health Organization to affect 350 million people worldwide . At presAutophagy is an important clearance system for cellular waste, including toxic protein aggregates . It is iIn addition to the autophagy pathways, many lines of evidence have implicated inflammation in the pathogenesis of depression and mechanisms of antidepressant response , 13. DepUp to now, it has been suggested that the role of innate and adaptive immunity could contribute to the development of MDD . MoreoveHowever, thus far, most investigations have been performed in cellular and animal studies with little progress being made in the clinical setting. Therefore, in this study, we measured serum levels of autophagy-related marker beclin-1 and some inflammatory markers  together at baseline in 40 patients with MDD. The primary aim of the current study was to analyze whether pre-treatment serum levels of beclin-1 and inflammatory factors could predict antidepressant treatment response. As a secondary aim, we investigated whether antidepressant treatment can change serum beclin-1 levels as there are no data in this regard.patients was consecutive. All diagnoses were made using the Structured Clinical Interview for DSM-IV (SCID) , venlafaxine/duloxetine. In this report, the study population was a subsample of the prospective randomized study cohort. Data on serum levels of beclin-1 and inflammatory factors were available from 40 MDD patients, because only 40 patients provided written informed consent for the biomarker study. The enrollment of V (SCID) –30. The All patients underwent a clinical interview, laboratory test, and physical examination to rule out physical illnesses. The patients were treated with SSRIs (paroxetine in 20 cases) or SNRIs (duloxetine in 13 cases and venlafaxine in 7 cases) for 8 weeks. The initial dose of paroxetine, duloxetine, and venlafaxine was 20, 40, or 75 mg/day, respectively, and the dose was increased according to the clinical response and patient tolerance (the dose was not fixed). The maximum dose of paroxetine, duloxetine, and venlafaxine was 40, 60, and 225 mg/day, respectively. The HAMD-17 was used to evaluate the severity of symptoms in MDD patients at baseline (w0) and after 8 weeks of treatment (w8). The protocol of this study was approved by the Ethics Committee of the Shanghai Mental Health Center. All patients gave written informed consent for the biomarker study. We defined the patients whose HAMD-17 scores decreased by 50% or more as responders and those with a less than 50% reduction as non-responders.Following an overnight fast, 5 ml of venous blood samples was collected between 7 and 9 a.m. After collection, the blood samples were centrifuged at 3,000 rpm at 4°C for 20 min and stored at −80°C before laboratory assays. The serum samples were collected both at baseline and after 8 weeks of antidepressant treatment.Serum levels of beclin-1, IL-1β, IL-6, and CRP for each sample were measured using the enzyme-linked immunosorbent assay (ELISA) method with a human beclin-1 ELISA kit , human IL-1β ELISA kit , human IL-6 ELISA kit , and high-sensitivity CRP ELISA kit  according to their respective manufacturer’s protocols. The concentrations of beclin-1, IL-1β, and IL-6 are expressed as pg of protein/ml of serum, and that of CRP is expressed as ng of protein/ml of serum. The detection limits for beclin-1, IL-1β, IL-6, and CRP were 15 pg/ml, 2 pg/ml, 7 pg/ml, and 10 ng/ml, respectively. The mean intra-assay and inter-assay coefficients of variation were below 9.6%.t-test for normally distributed continuous variables and the Mann–Whitney U test for non-normally distributed variables. The chi-square test or continuity-adjusted chi-square test was used for dichotomous variables, when appropriate. The Mann–Whitney U test was used to evaluate the significance of differences in individual serum biomarker baseline levels expressed between the responder group and the non-responder group. To investigate whether baseline serum factors level could be used to predict an antidepressant treatment response in patients, the following statistical methods were used.Baseline clinical characteristics were compared between the responder and non-responder groups using unpaired First, we used the logistic regression model. The univariate logistic regression analysis was performed to examine the predictive potential of serum factors. Then, to assess any independent factors affecting the response to antidepressant treatment, multivariate logistic regression analysis was performed for variables with a statistically significant difference in the univariate logistic regression (p < 0.1). To avoid omitting significant predictors, we included some important sociodemographics and clinical characteristics and constructed several multiple forward logistic regression models. Multicollinearity was examined using collinearity diagnostic statistics. Variance inflation factor (VIF) values > 4.0 or tolerance <0.25 may indicate a concern for multicollinearity in multivariate regression models . The Boxjp2( j∣t), where p( j∣t) is the estimated class probability for feature t or node t in a decision tree and j is an output data or class. In this study, j = 2 is represented as responders = yes and responders = no  for the contribution to predicting antidepressant response. The Gini index is commonly used to measure the ability of a potential discriminative test of each feature that can be defined as 1 − ∑ers = no . The inpers = no .p-value of <0.05 was considered statistically significant. Statistical analyses were performed using SPSS 17.0  software and R programming language (version 3.2.2 for Windows).All results are presented as the mean ± standard deviation. Statistical tests were two tailed, and a Z = −5.375, p < 0.001). Of all patients, 27 (67.5%) patients were responders and 13 patients were non-responders. The demographic characteristics and clinical data of responder patients and non-responder patients are listed in A total of 40 subjects  were included in the analysis. After 8 weeks, all patients completed the follow-up evaluation and sample collection. Treatment for 8 weeks resulted in significant improvement of depressive symptoms as measured by HAMD-17 total scores . No significant differences in the levels of serum IL-1β, IL-6, or CRP were detected between non-responders and responders . There were no significant correlations between baseline levels of beclin-1, IL-1β, IL-6, and CRP with age, sex, BMI, or baseline HAMD-17 total scores—neither in the total sample nor in the two study groups (data not shown). Moreover, there were no significant correlations between baseline levels of beclin-1 and baseline IL-1β, IL-6, and CRP levels—neither in the total sample nor in the responder and non-responder groups (data not shown).As shown in p = 0.01], while serum IL-1β, IL-6, and CRP level were not significantly correlated with treatment response   (p = 0.001) , univariate logistic regression analysis showed that the beclin-1 level significantly negatively correlated with treatment response [OR = 0.993, 95%  confidence interval (CI) 0.988 to 0.998, p > 0.1) Table 2.To further support our results, we used a random forest model to sort the order of importance of these serum-biomarker and demographic data in terms of their contribution to the treatment response. As shown in Z = −1.290, p = 0.197). Further sub-group analysis showed that the serum levels of beclin-1 were significantly increased in the responders group whereas no change was detected in the non-responder group . No significant correlation was found between change in serum beclin-1 level and reduction in HAMD-17 scores in all patients  or each patient subgroup  following treatment.We compared pre-treatment and post-treatment of serum beclin-1 levels to determine whether antidepressant treatment could change serum beclin-1 levels. No significant change in serum beclin-1 was observed in all patients after 8 weeks of antidepressant treatment  and citalopram (an SSRI) could increase the expression of beclin-1 in rat primary astrocytes, but venlafaxine fails to exert these effects. Thus, some but not all antidepressants could affect autophagy . In other words, serum beclin-1 may not be used to predict response to a specific type of antidepressants. Thus, beclin-1 may not be used to guide the choice of antidepressants but rather to identify patients that potentially may be helped by early adjuvant therapies. Future studies with a large sample sizes are needed to further clarify whether there could be a differential effect of pre-treatment beclin-1 levels on treatment outcomes with SSRI vs. SNRI. Third, we have not investigated potential biological mechanisms underlying the association of low serum beclin-1 level with a good response. The exact mechanism still needs further study. Fourth, whether beclin-1 could change after 8 weeks of antidepressant treatment in other populations and the time taken for beclin-1 levels to change remain unknown. Further studies focusing on other populations are required. Fifth, other autophagy markers, such as LC3B-II/I and p62, have not been systematically investigated in this study. It is necessary to study other markers in the future. Finally, the cellular source of serum beclin-1 remains unclear, and whether the serum levels of beclin-1 reflect the real levels in the CNS cannot be concluded from the present study. In conclusion, despite the aforementioned limitations, the findings of our study suggest that baseline serum beclin-1 levels may be used as a biomarker to predict antidepressant response in patients with MDD. Moreover, our results provide further support that beclin-1 may be involved in the therapeutic effect of antidepressant drugs.The protocol of this study was approved by the Ethics Committee of the Shang Hai Mental Health Center. All patients gave informed consent for the biomarker study.Author SH performed the statistical analyses and wrote the manuscript. Author DZ and ZL provided assistance for the laboratory work. Author JS completed all of the data entry. Authors FX, NZ and QW were responsible for the diagnosis and clinical assessment of the participants. Author HL designed and wrote the study protocol and reviewed the manuscript. In addition, author JZ and WY offered many constructive opinions on this study and provided a critical revision of the manuscript for important intellectual content. All authors contributed to and approved the final manuscript.This study was supported from the Collaborative Innovation Center for Translational Medicine at Shanghai Jiao Tong University School of Medicine (TM201506 and TM201624); Clinical Research Center, Shanghai Jiao Tong University School of Medicine (DLY201620); Youth Scientific Research Project of Shanghai Municipal Commission of Health and Family Planning (20184Y0310); and National Major Project for IND (2018ZX09734005).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


D-stereospecific proteases (DSPs) in resolution of racemic amino acids and in the semisynthesis of proteins has been a successful strategy. The main limitation for a broader application is, however, the accessibility of suitable DSPs covering multiple substrate specificities. To identify DSPs with novel primary substrate preferences, a fast specificity screening method using the easily accessible internally quenched fluorogenic substrate aminobenzoyl-D-arginyl-D-alanyl-p-nitroanilide was developed. By monitoring both UV/vis-absorbance and fluorescence signals at the same time it allows to detect two distinct D-amino acid substrate specificities simultaneously and separately with respect to the individual specificities. In order to identify novel DSP specificities for synthesis applications, DSPs specific for D-arginine were of special interest due to their potential ability as catalysts for substrate mimetics-mediated peptide and protein ligations. D-alanine in the substrate served as positive control and reference based on its known acceptance by numerous DSPs. In silico analysis suggested that DSPs are predominantly present in gram-positive microorganisms, therefore this study focused on the bacilli strains Bacillus thuringiensis and Bacillus subtilis as potential hosts of D-Arg-specific DSPs. While protease activities toward D-alanine were found in both organisms, a novel and so far unknown D-arginine specific DSP was detected within the culture supernatant of B. thuringiensis. Enrichment of this activity via cation exchange and size exclusion chromatography allowed isolation and further characterization of this novel enzyme consisting of a molecular mass of 37.7 kDa and an enzymatic activity of 8.3 U mg-1 for cleaving the D-Arg|D-Ala bond in the detecting substrate. Independent experiments also showed that the identified enzyme shows similarities to the class of penicillin binding proteins. In future applications this enzyme will be a promising starting point for the development of novel strategies for the semisynthesis of all-L-proteins.The application of  This similarity makes OGp esters perfect substrates for arginine-specific proteases. Furthermore, the direct use of D-amino acids within the sequence of antimicrobial agents or for pharmaceutical applications is also widely considered (DD-peptidases (DSPs) (E.C. 3.4.16.4), originally catalyzing the hydrolysis of a peptide bond between two D-amino acid moieties, would have a significant potential as ligation catalyst in the semisynthesis of those biopolymers.Proteases are ubiquitously distributed in all three domains of life and fulfill functions in various cellular and biological processes, nutrient acquisition and microbial defense processes . In addi by DSPs . By thisnsidered . HoweverDSPs relates to the limited range of distinct substrate specificities of these enzymes. Previously, D-amino acid accepting enzymes like D-aminoacylase, D-aminopeptidase or the ADP were described 4 and classical FRET (Förster Resonance Energy Transfer) substrates  with the dual use as chromogenic substrate was evaluated. In addition the intended substrate allows a site-specific determination of hydrolytic events toward a distinct amino acid without the need of an ongoing MS analysis step of the peptides substrate. Classical IQFSs normally consist of a fluorescence donor group at one terminal end of the substrate and a quenching acceptor counterpart at the other  located at the C-terminal end of the substrate . At the same time, analysis of the liberated pNA group via simultaneous vis-spectroscopy (λ 405 nm) enables the detection of enzyme activities toward D-Ala exclusively. In this case study we used the advantages of robustness, sensitivity, and specificity of the approach to identify and isolate novel D-arginine specific hydrolases.Keeping the already proven enzymatic activities for the cleavage of tability . Due to bstrates , 2013. Ghe other . The genroteases . In thisubstrate . In addi® Protein & Peptide C18) via gradient elution with water/acetonitrile (v/v) ranging from 20 to 80% ACN containing 0.1% trifluoroacetic acid at room temperature and flow rates of 10 ml min-1. Product elution was followed at 220 nm. The overall product yield of the whole synthesis was 36%. The identity and purity of the product was determined by mass spectrometry  and analytical HPLC at 220 nm, respectively (see enantiomeric excess (ee) of the final peptide derivative was found to be higher 99% as proven by enzymatic resolution experiments using the L-Ala and L-Arg specific proteases elastase and trypsin, respectively. Resolution reactions were performed with 2 mM of the IQFS and 5 μM of the respective protease for 4 h. Within the detection limit of the fluorescence measurements, which determine concentrations ≥0.01 μM, no cleavage products could be detected indicating an ee higher than 99% for the IQFS synthesized. Analog reactions with the corresponding all-L-IQFS were performed as positive control resulting in a complete hydrolysis of the substrate.Amino acid derivatives, coupling reagents, media components, and chemical compounds were obtained from Bachem (Switzerland), Merck (Germany), Roth (Germany), and Fluka (Germany). All reagents were of the highest commercial purity. If necessary, solvents and reagents were purified and/or dried by standard procedures. The synthesis of the internally quenched fluorescence substrate was performed by standard coupling, purification and deprotection methods as depicted in vely see . The enaBacillus thuringiensis Berliner 1915 and Bacillus subtilis subsp. subtilis were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). Bacillus strains were grown in 50 ml NB-medium in 250 ml flasks under continuous shaking for 24 h at 30°C  for cell disruption and later analyzed as “culture fraction” reflecting the complete DSP activity originated by the cells. Remaining cells were sedimented at 4,000 ×g for 15 min, yielding 0.13 and 0.21 g/10 ml wet weight for B. subtilis and B. thuringiensis, respectively. The “culture supernatant fraction” was separated via decantation and the cells (‘whole cell fraction‘) were resuspended in ml resuspension buffer . After incubating the suspension for 30 min at 37°C, the cells were spun down for 10 min at 10,000 ×g resulting in a two phase supernatant and a pellet. The upper phase of the supernatant was removed, representing the “cell wall fraction” and the remaining supernatant was discarded. The pelleted cells were resuspended in 1 ml of cold PBS and lysed by sonication (10 s pulses at 40% amplitude). After two further washing/centrifugation steps at 50,000 ×g for 30 min at 4°C and at 400,000 ×g for 45 min at 4°C the top 30% of the supernatant were kept as “cytoplasmic fraction.” The pellet was washed twice in PBS and centrifuged at 400,000 ×g for 45 min at 4°C. The suspended pellet in PBS was kept as “membrane fraction.” For a better comparability of the results, an amount corresponding to 1 mg wet weight of the original whole cell fraction paste was used within the initially activity measurements.The gram-positive bacterial strains  at 30°C . 100 ml Bacillus thuringiensis was grown in a total volume of 14 liters of NB-medium with continuous shaking at 30°C for 24 h. After separating the cells by centrifugation at 5,000 ×g for 30 min, the supernatant proteins were enriched using a two-step chromatographic strategy at 8°C using phosphate buffer . For this purpose, the supernatant was loaded on a Toyopearl® Supelco SP 650 M column and eluted with a single step gradient to 0.4 M NaCl. Fractions containing the desired enzymatic activity were identified using IQFS, pooled, concentrated, and dialyzed against the original phosphate buffer. The last step of purification represents a size exclusion chromatography . D-Arg DSP containing fractions were combined, concentrated, and analyzed via SDS-polyacrylamide gel electrophoresis and mass spectrometry.DSPs in putative host organisms was carried out using the database eggNOG3.0 . Elution was performed with an acetonitrile/water gradient from 0 to 40% (v/v) ACN containing 0.05% trifluoroacetic acid at a flow rate of 0.5 ml min-1. Multiple wavelengths for detection were used simultaneously . For LC-MS analysis a RP C18 column (X-BridgeTM BEH300), gradient elution and a Waters micromass ZQ detector were used.Enzyme activities were determined by measuring the rate of hydrolysis using a NOVOstar microplate reader  and clear-bottom 96-well plates . For fluorescence analysis an excitation wavelength of 320 nm and an emission wavelength of 420 nm were used. Simultaneously, released 5 nm see . All meaDP-like DSPs with alternative D-amino acid specificities, bioinformatic homology studies were initially performed  compared to that corresponding to the absorption (217 mU g-1). From this disparity one can conclude a bias in the substrate specificity toward the cleavage after D-Arg over D-Ala. In contrast, no difference between both signals was found for B. subtilis (fluorescence-based activity 219 mU g-1 vs. absorption-based activity 203 mU g-1). Analysis of the whole cell fraction of B. subtilis  and the membrane (74 mU g-1). Related to the latter, nearly the same DSP-activity was found within the membrane fraction of B. thuringiensis (69 mU g-1), while the cell wall fraction showed less DSP-activity compared to B. subtilis (22 mU g-1). Strikingly, the most significant differences between the fluorescence- and absorbance-based activities  arise for the supernatant fraction of B. thuringiensis. This strongly indicates a unique D-Arg specific DSP, which is obviously not present in B. subtilis, accounting for the bias of both signals in the B. thuringiensis strain. To confirm the enzymatic cleavage of the D-Arg-D-Ala-bond as indicated by the dominant fluorescence signal, additional HPLC-MS analyses of the reaction mixture were performed   .D-stereospecific Proteases still represent an underestimated class of proteases with only a very limited variety of distinct D-amino acid specificities. On the other hand, a significant quantity of applications in academic as well as applied research has been already developed which are, however, hampered by the only small number of suitable enzymes presently known. To broaden the scope of these interesting enzymes in this context, identification of novel DSPs with alternative D-amino acid specificities is an important approach. Regarding our own concerns, DSPs specific for D-Arg are of particular interest especially for their use as ligation catalysts in the synthesis and modification of native all-L-peptides and -proteins. DSPs with D-Arg specificity are, however, unkown so far. To avoid overlapping with a second, undesired substrate specificity, a novel dual IQFS substrate was developed enabling the analysis of two distinct amino acid specificities at the same time in a fast, robust and sensitive manner. Although classical IQFSs are already well established especially for the characterisation of L-stereospecific proteases 4 or (D-Asp)8 or in combination with substrates bearing single chromogenic functionalities (DSPs mainly for profiling DSPs in order to classify organisms (Bacillus spp. have the potential to cleave substrates consisting of two D-amino acids such as D-Lys-D-Lys, D-Leu-D-Leu and D-Arg-D-Arg (B. thuringiensis within hours, the used IQFS was also utilized to isolate and finally characterize novel DSP. Remarkably the last step can be performed within minutes and demonstrating the high potential of this substrate class for characterizing DSPs. In contrast, the dual IQFS used in our study has the advantage to screen for at least two distinct substrate specificities in only one single screening step simultaneously. Beside its sensitifity, flexibility and simplicity in application, the use of the substrate is rather pH independent enabling screenings at variuos pH-values ranging from pH 4 to 9 (DSPs due to occurrence of alternative metabolism pathways or D-amino acid containing peptides and proteins in the microbiome.roteases , we coulnalities . Classicrganisms . Besidesrg-D-Arg . Whilst H 4 to 9 . Based oDSPs specific for D-Ala within the investigated organisms can be correlated to the occurrence of PrPs and PbPs within that group of Bacilli, which are already known to exhibit D-Ala protease activity (Bacilli (Bacilli strains used were fractionated in their late exponential growth phase going along with the finding that the highest D-Ala proteolytic activity was detected in the membrane and cell wall fractions. In contrast, the additional proteolytic activity toward D-Arg was found in the supernatant of the culture of B. thuringiensis indicating a biological function of the novel DSP distinct from classical PrPs and PbPs. Interestingly, D-aminopeptidase from O. anthropoi as well as ADP from B. cereus, two enzymes with lower similarity to PrPs and PbPs, were also isolated from the supernatant of the respective cell cultures (D-Arg specific DSP adds a novel D-amino acid specificity to the known repertoire of D-stereospecific proteases which might be suitable for application in protein semisynthesis or racemic resolution. Studies in this direction are presently underway.The detection of activity . It is f(Bacilli . Keepingcultures . From a TA, AS, and SL conceived of the present idea. TA and AS carried out the chemical synthesis. Bioinformatic studies and biological assays were carried out by AS. All authors discussed the results and contributed to the final manuscript. AS, TA, and SL wrote the manuscript with support from FB. FB supervised the project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of the present study was to investigate the effects of different routes of bvPLA2 administration in a PD mouse model. Neurobehavioral assessment revealed progressive deterioration in locomotor functions of the MPTP group compared with the control group. However, such functions were improved following subcutaneous (s.c.) bvPLA2 administration. The results showed that the s.c. route of bvPLA2 administration contributed to the induction of Treg cells and the reduction of Th1 and Th17 populations, demonstrating that the neuroprotective effects were associated with reduced tyrosine hydroxylase (TH)-positive dopaminergic neurons and microglia. These results suggested that the s.c. bvPLA2 injection could be beneficial for treating aspects of PD.Parkinson’s disease (PD) is the second most common neurodegenerative disorder worldwide. Progressive loss of dopaminergic neurons in the substantia nigra (SN) and their synaptic terminal connections in the striatum are main characterizations of PD. Although many efforts have been made to develop therapeutics, no treatment has been proven effective. We previously demonstrated that bvPLA2 can protect dopaminergic neurons by modulating neuroinflammatory responses in an MPTP -induced mouse model of PD. The cellular basis for the neuroprotective response of bvPLA2 was the induction of CD4 Tregs mediate neuroprotection through the modulation of inflammatory responses, suppression of microglial activation, and promotion of neuronal survival in animal models of PD  is a common degenerative disease of the central nervous system that affects motor functions, such as muscular rigidity, onset of tremor, slowness of voluntary movements and difficulty maintaining balance. PD is caused by the progressive loss of dopaminergic neurons in the substantia nigra (SN). Current symptomatic therapy for PD can provide a benefit to slow disease progression and may cause motor complications, such as motor fluctuation and dyskinesia. Therefore, therapeutic strategies to delay the onset or slow progression of PD are needed. Functional links between adaptive immunity and neurodegenerative disorders have been well studied for PD, Alzheimer’s disease (AD) and multiple sclerosis  extracted from honeybees is commonly used in Korean medicine. Previous studies have reported that BV therapy has anti-nociceptive and anti-inflammatory effects on pain, arthritis, cancer and skin disease , subcutaneous (s.c.), intramuscular (i.m.) and intravenous (i.v.) injection routes were examined. The findings highlight the optimum route of bvPLA2 administration to exert its neuroprotective effects on MPTP-induced PD in a mouse model by controlling the generation of Treg populations.BV contains several bioactive compounds, including melittin, phospholipase A2 (PLA2), apamin and peptides  were purchased from The Jackson Laboratory . The mice were maintained on a 12 h light/dark cycle and temperature-controlled conditions (21 ± 2°C), with food and water For MPTP intoxication, the mice received four i.p. injections of MPTP-HCl  at 2-h intervals as previously described  facing upwards. The animals were allowed to climb down to the base of the pole. The time to turn completely downward (T-turn) and the total time taken for the mouse to reach the floor (locomotion activity time [T-LA]) were recorded, with a cut-off limit of 30 s. Each mouse was examined in five trials, and the average of the best three measurements was calculated. Trials with the mouse jumping or sliding down the pole were excluded.6 cells/ml were collected, washed twice with phosphate-buffered saline (PBS), and stained with FITC-conjugated anti-CD4 and PE-labeled anti-CD25 antibodies in staining buffer for 30 min on ice in the dark. The cells were subsequently washed twice with PBS and fixed in fixation/permeabilization buffer (eBioscience) for 1 h at 4°C in the dark. Subsequently, the cells were washed twice with PBS and stained with Alexa Fluor 647 anti-Foxp3 antibody overnight at 4°C in the dark. After washing, the cells were fixed in 1% paraformaldehyde and stored at 4°C in the dark for subsequent detection. For intracellular cytokine staining, single-cell splenocytes at a concentration of 2 × 106 cells/ml were stimulated with 50 ng/ml of phorbol myristate acetate  and 1000 ng/ml of ionomycin (Sigma-Aldrich) for 1 h, and then incubated with GolgiStop (BD Biosciences) for 4 h. The cells were collected, washed twice with PBS, and stained with FITC-labeled anti-CD4 antibody for 30 min on ice in the dark. After washing with PBS, the cells were fixed in IC fixation buffer (BD Biosciences) for 30 min at room temperature in the dark. The cells were then stained with PE-conjugated anti-IFN-γ, PE-conjugated anti-IL-4 and PerCP-Cyanine5.5-conjugated anti-IL-17A antibodies overnight at 4°C -conjugated anti-mouse CD4 , phycoerythrin (PE)-conjugated anti-mouse CD25 , Alexa Fluor 647 anti-mouse/rat Foxp3 , PE-conjugated anti-mouse IFN-γ  PE-conjugated anti-mouse IL-4 , and PerCP-Cyanine5.5-conjugated anti-mouse/rat IL-17A  antibodies. For Treg staining, single-cell splenocytes at a concentration of 2 × 10The mice were anesthetized with isoflurane  and transcardially perfused with PBS followed by perfusion with a fixative solution containing 4% paraformaldehyde dissolved in 0.1 M phosphate buffer. The brain was dissected, postfixed in 4% paraformaldehyde at 4°C overnight, transferred to 30% sucrose solution, and subsequently frozen. The tissues were embedded in OCT compound and serially cut on a cryostat into 30-μm thick coronal sections by using a sliding microtome. All sections were collected in six separate series and processed for immunohistochemical staining. Primary antibody was directed against tyrosine hydroxylase  and Iba-1 (1:2000), Wako Pure Chemic Industries, Osaka, Japan). After washing with PBS, the sections were incubated with the appropriate biotinylated secondary antibody and processed with an avidin-biotin complex kit . The reaction product was visualized with 0.05% diaminobenzidine-HCL (DAB) and 0.003% hydrogen peroxide in 0.1 M phosphate buffer. The labeled tissue sections were subsequently mounted and analyzed under a bright field microscope .Unbiased stereological estimation of the total number of TH-positive dopaminergic neurons in the SN was performed by using an optical fractionator method on an Olympus CAST  system version 2.1.4.  as previously described . The data are presented as the means ± standard error of the mean (SEM) where indicated. Statistical significance of each variable was evaluated by one-way analysis of variance (ANOVA), followed by Newman-Keuls multiple comparison test for multiple comparison or by two-tailed Student’s To evaluate the effects of different routes of bvPLA2 administration on MPTP-induced neurotoxicity, mice were administered MPTP and received a single daily injection via i.p., s.c., i.m. and i.v. of bvPLA2 or saline for 6 days commencing 12 h after the last MPTP injection. Various behavioral tests have been routinely used to qualify PD mouse models, including locomotor activity, rotarod test, forepaw stride length, and the pole test  is an effective and potent neuroprotective agent for PD. However, GDNF drug development has failed, because the neurotrophic factor does not cross the blood-brain barrier (BBB; Bartus and Johnson, +CD25+Foxp3+ Treg populations. We suggested that the neuroprotective effects of bvPLA2 treatment in MPTP-induced PD model resulted from the increased infiltration of memory T lymphocytes through choroid plexus instead of direct across the BBB.Over the past few decades, many efforts have been made to identify new drug targets focusing on CNS disease, including AD, PD and Huntington disease (Trojanowski and Hampel, + T cells isolated from Cop-1 immunized mice evoked a robust neuroprotective response (Laurie et al., + T cells in protective responses in PD. Furthermore, Reynold et al. have reported that the adoptive transfer of CD4+CD25+ Tregs can modulate immune responses in the brain, resulting in significant neuroprotection in a PD mouse model (Reynolds et al., A growing body of evidence suggests that cellular and humoral immune responses are changed in the peripheral immune system of PD patients (Dauer and Przedborski, In PD, autoreactive Th1 or Th17 cells are important for the promotion of neurodegeneration. A shift in the Th1/Th2 balance toward Th1 is closely associated with motor function. Th17 cells play critical roles in the protection against mucosal inflammation. Recent studies on the neurotoxic properties of Th17 populations have reported that the infiltration of Th17 cells into the SN can exacerbate dopaminergic neuronal loss in MPTP intoxication models (Liu et al., The present study reported an optimized route of bvPLA2 administration to prevent neuronal damage in an MPTP-induced PD mouse model. The s.c. injection of bvPLA2 represented the best route of administration among i.p., s.c., i.m., and i.v. injections. The results from the present study suggest that the s.c. route of bvPLA2 administration might have potential application for the treatment of PD patients.HNJ and HIJ performed the experiments and conducted the statistical analysis. HBaek drafted the first version of the manuscript. HNJ and HBaek drafted sections of the manuscript. HBae supervised the project and edited the manuscript. All authors contributed to the manuscript revision and have read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Introduction: Excessive co-contraction interferes with smooth joint movement. One mechanism is the failure of reciprocal inhibition against antagonists during joint movement. Reciprocal inhibition has been investigated using joint torque as an index of intensity during co-contraction. However, contraction intensity as an index of co-contraction intensity has not been investigated. In this study, we aimed to evaluate the influence of changes in contraction intensity during co-contraction on reciprocal inhibition.Methods: We established eight stimulus conditions in 20 healthy adult males to investigate the influence of changes in contraction intensity during co-contraction on reciprocal inhibition. These stimulus conditions comprised a conditioning stimulus-test stimulation interval  of -2, 0, 1, 2, 3, 4, or 5 ms plus a test stimulus without a conditioning stimulus (single). Co-contraction of the tibialis anterior and soleus muscles at the same as contraction intensity was examined at rest and at 5, 15, and 30% maximal voluntary contraction (MVC).Results: At 5 and 15% MVC in the co-contraction task, the H-reflex amplitude was significantly decreased compared with single stimulation at a 2-ms C–T interval. At 30% MVC, there was no significant difference compared with single stimulation at a 2-ms C–T interval. At a 5-ms C–T interval, the H-reflex amplitude at 30% MVC was significantly reduced compared with that at rest.Discussion: The findings indicated that during co-contraction, reciprocal Ia inhibition worked at 5 and 15% MVC. Contrary inhibition of reciprocal Ia inhibition did not apparently work at 30% MVC, and presynaptic inhibition (D1 inhibition) might work. In many upper motor neuron disorders including spastic diseases, cerebellar ataxia, Parkinson’s disease, and spinal cord injury, when movement of only an agonist is required , a collapse of the reciprocal inhibition mechanism against the antagonist causes excessive co-contraction. Thus, reciprocal inhibition does not work on the antagonist, impairing smooth joint movement . Such exA previous study on reciprocal inhibition during co-contraction of the tibialis anterior (TA) and soleus (Sol) muscles in healthy men reported that the amount of reciprocal Ia inhibition was further decreased during co-contraction than at rest . In thatThis study thus aimed to clarify the influence of changes in contraction intensity during co-contraction on reciprocal inhibition. Other researchers e.g.,  have repTwenty healthy adult males  provided written informed consent to participate in this study. The study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee at Niigata University of Health and Welfare.The right lower limb position was measured at the hip (90°), knee 120°), and ankle (90°) joints. The ankle was immobilized using a foot plate   of the surface electromyogram was set to 20 mm. The electrodes were placed on the TA and Sol muscles according to SENIAM . The groMuscles were stimulated for 1 ms (rectangular wave) using a SEN-8203 electrical stimulation device  via a SS-104J isolator (Nihon Kohden). The tibial nerve was selectively stimulated in a monopolar fashion to induce the Sol H-reflex and M waves. The anode and cathode were located on the upper patella and the popliteal area, respectively, for the test stimulus. M waves were induced in the TA using bipolar stimulation, and a conditioning stimulus was applied along the deep peroneal nerve below the fibula head .Reciprocal inhibition was measured as described previously . A test The contraction intensities of TA and Sol were determined by measuring the maximal voluntary contraction (MVC). MVC was performed for plantar- and dorsiflexion with the thighs and feet fixed as shown in Figure The experimental procedure is shown in Figure Post hoc Tukey–Kramer multiple comparison tests were then performed. The seven stimulation conditions were compared with the single condition using paired t-tests with the Bonferroni correction for multiple comparisons. Co-contraction tasks under each stimulation condition were compared using two-way repeated-measures ANOVA with the factors, co-contraction task, and stimulation condition, followed by post hoc Tukey–Kramer multiple comparison tests. Statistical significance was set at p < 0.05.Peak-to-peak Sol H-reflex amplitudes were calculated as mean ± standard error (SE). The reciprocal inhibition of each co-contraction task was calculated as a percentage by dividing the Sol H-reflex amplitude by the Mmax amplitude ([Sol H-reflex amplitude/Mmax amplitude] × 100). To compare the amount of reciprocal inhibition between simultaneous contraction tasks under the seven stimulation conditions (excluding the single condition), the H-reflex amplitude for the conditioning stimulus was divided by the H-reflex amplitude of only the test stimulus and expressed as a percentage ([Conditioned H-reflex amplitude/Test H-reflex amplitude] × 100). Values are expressed as mean ± standard deviation. Iterative values were generated using two-way repeated-measures analysis of variance of the factors, co-contraction task, and stimulation conditions. F = 36.271, p < 0.001, partial η2 = 0.656], but not of the co-contraction task . In addition, the stimulation condition and the co-contraction task interacted significantly . Analyses of the single Sol H-reflex amplitudes during each co-contraction task and multiple comparisons did not identify any significant differences. These findings confirmed that changes in the Sol H-reflex amplitude in response to conditioning stimuli do not depend on the test stimulus intensity.In the two-way repeated measures ANOVA of the co-contraction task and stimulation conditions, there was a main effect of the stimulus condition  of each stimulation condition but not that of the co-contraction task . Additionally, the stimulation condition and co-contraction task interacted significantly . Multiple comparison tests, followed by post hoc analyses, did not identify any significant differences between co-contraction tasks at C–T intervals of -2, 0, 1, or 2 ms. In the co-contraction task at 30% MVC at a C–T interval of 4 ms, the H-reflex amplitude was significantly reduced compared with that at rest (p = 0.009) and at 5% MVC (p = 0.014). In the co-contraction task at 30% MVC with a C–T interval of 5 ms, the H-reflex amplitude was significantly reduced compared with that at rest (p < 0.001) as well as that at 5% (p < 0.001) and 15% MVC (p = 0.045).Figure The main findings of the present study are that reciprocal Ia inhibition occurred at 5 and 15% MVC due to changes in contraction intensity during co-contraction at the same intensity between the TA and Sol muscles. In addition, we demonstrated that the amount of reciprocal Ia inhibition during co-contraction of 5 and 15% MVC was the same as that at rest. Reciprocal Ia inhibition was not evident at 30% MVC, suggesting D1 inhibition.Our findings at rest were similar to those of a previous study , which sDuring co-contraction at 5 and 15% MVC, the Sol H-reflex amplitude was significantly decreased at a C–T interval of 2 ms compared with the single condition. These findings are similar to our hypothesis, as reciprocal inhibition differed from previous findings, and the extents of reciprocal Ia inhibition at 5 and 15% MVC were the same as those at rest. Previous studies have investigated changes in the amounts of reciprocal Ia inhibition during co-contraction at the same joint torque during plantar- and dorsiflexion . By contAt 30% MVC for the single condition and C–T intervals at 2 ms, the Sol H-reflex amplitude did not differ significantly. This result suggests that reciprocal Ia inhibition decreases in the Sol muscle depending on its contraction intensity. Excessive simultaneous contraction intensity also decreases joint movement . The intThe H-reflex amplitude was significantly decreased at 30% MVC compared with that at rest at C–T intervals of 4 and 5 ms. Previous studies have reported the involvement of D1 inhibition in the inhibition mechanism at C–T intervals of 5 ms or longer . D1 inhiOne limitation of this investigation is that it studied reciprocal inhibition at a middle latency of 3 to 5-ms C–T intervals. In reciprocal inhibition, short latency is reciprocal Ia inhibition (2 ms) and long latency is D1 inhibition (5–80 ms) . The repReciprocal Ia inhibition under changes in contraction intensity during co-contraction is yet to be fully elucidated. The present study confirmed reciprocal Ia inhibition at TA and Sol co-contraction at 5 and 15% MVC at the same contraction intensity. During co-contraction at 5 and 15% MVC, antagonist inhibition may occur during joint movement. However, co-contraction at 30% MVC decreased reciprocal Ia inhibition, suggesting that joint fixation could be affected. The influence of changes in co-contraction strength on reciprocal inhibition at intermediate latency requires further investigation, and suppression during co-contraction should be studied by combining contraction intensity with joint torque. Future studies should also investigate the influence of changes in co-contraction intensity on the reciprocal inhibition of middle latency, as well as reciprocal inhibition during co-contraction, by combining contraction intensity and joint torque.RH designed the study and wrote the initial draft of the manuscript. ME, SK, and HO contributed to analysis and interpretation of data and assisted in the preparation of the manuscript. All other authors have contributed to data collection and interpretation and have critically reviewed the manuscript. All authors approved the final version of the manuscript and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this work, the effect of doubly functionalized montmorillonite (MMT) on the structure, morphology, thermal, and tribological characteristics of the resulting polystyrene (PS) nanocomposites was investigated. The modification of the MMT was performed using a cationic surfactant and an anionic surfactant or a silane coupling agent to increase the compatibility with PS matrix. The polystyrene/organo-montmorillonite (PS/OMMT) nanocomposite particles were prepared by soap-free emulsion polymerization. The OMMT was studied using Fourier-transform infrared (FTIR), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). The structural and morphological changes of PS/OMMT nanocomposites were further characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The thermal stability of all the PS/OMMT nanocomposites was higher than that of the pure PS. The anti-wear properties of the polyalphaolefin (PAO) were significantly improved due to the introduction of the PS/OMMT nanocomposite particles. The nanocomposites prepared by a cationic surfactant and a silane coupling agent exhibited the best thermal stability and tribological performance. Our results provide the valuable insights needed to guide the design of lubrication and friction reducing materials. Therefore, the natural state of the MMT was a hydrophilic character, which was inherently incompatible with the vast majority of organic polymers [Clay-polymer nanocomposites have received great attention in recent years due to their exceptional properties, such as increased thermal stability, high fire resistance, and enhanced mechanical characteristics, compared with traditional polymer composites ,2. The i the MMT . To neutpolymers . The poopolymers . The surThe most traditional and convenient method for the MMT modification was ion exchange reaction with quaternary alkylammonium salts . HoweverPolystyrene (PS) is a synthetic thermoplastic that is widely used in industry due to its excellent performance of high abrasion resistance, strong plasticity and load bearing capacity . HoweverIn this work, two kinds of doubly organo-modified MMT were successfully prepared by using both a cationic surfactant and an anionic surfactant or both a cationic surfactant and a silane coupling agent. Then, the PS/OMMT nanocomposites were produced using styrene monomer and the obtained OMMT samples via the soap-free emulsion polymerization. The obtained PS/OMMT nanocomposites were dispersed into polyalphaolefin (PAO) to create lubricants. The effect arising from the doubly organo-modified MMT on the structure, morphology, thermal, and tribological properties of the PS/OMMT nanocomposites were studied and evaluated. The natural sodium montmorillonite  of 100 mmol/100 g) was provided by Huai An Saibei Technology Co. Ltd., Zhangjiakou, China. The tributyltetradecylphosphonium chloride , sodium lauryl sulfonate , and γ-methacryloxypropyltrimethoxysilane  were purchased from Aladdin Industrial Corporation, Shanghai, China. Styrene , divinylbenzene , methacrylic acid , and potassium persulfate  were supplied by the Tianjin Guang fu Fine Chemicals Research Institute, Tianjin, China. The St was washed with NaOH solution to remove the polymerization inhibitors, and then distilled under reduced pressure before use. Polyalphaolefin  was provided by Shanghai Fox Chemical Technology, Shanghai, China. Other reagents were of analytical grade and used as received. Deionized water was applied for all experiments.The modification of MMT with TTPC was carried out by the method reported in a previous paper . BrieflyThe P-MMT was further modified using the SLS surfactant to produce the doubly organo-modified MMT (P-SLS-MMT) as shown in The silane functionalization of P-MMT was carried out following the method described in a previous report . In a tyThe soap-free emulsion polymerization was applied to prepare the PS/OMMT nanocomposites according to previous reports ,25. BrieThe PAO-PS/OMMT lubricant was prepared to investigate the tribological performance of the PS/OMMT nanocomposites as additives. The PS/OMMT nanocomposites (1.0 wt % based on PAO) were added to the PAO, and the mixture was mechanically stirred at 1000 rpm for 30 min followed by ultrasonication for 1 h at room temperature to allow the dispersion and homogenization of the nanocomposites in the PAO. −1 using an FTS-3000 spectrophotometer (American Digilab) via a KBr pressed disk technique at room temperature. Fourier-transform infrared (FTIR) spectra were conducted over the range of 4000–400 cmX-ray diffraction (XRD) measurements were carried out with a Bruker D8 Advance X-ray Diffractometer  using Cu-Ka radiation (λ = 0.1540 nm) with a scanning rate of 2°/min, 40 kV, and 30 mA over the 2θ range of 1.5°–20° by a scanning interval of 0.02°. The corresponding basal space was computed by applying the Bragg's Law. Dynamic light scattering (DLS) was performed using a Malvern Mastersizer 2000  to determine the particle size and size distribution of the samples at room temperature. Scanning electron microscopy  coupled with an energy dispersing spectrometer (EDS) was used to study the surface morphology and elements of the nanocomposites. The samples were coated with a thin layer of gold prior to SEM observation. The EDS analysis was registered three times in different points, and the result was the average of three obtained values. Transmission electron microscopy (TEM) analyses were performed on a JEM-2100 TEM operating at an accelerating voltage of 200 kV. Thermogravimetric analysis (TGA) was carried out on a NETZSCH thermogravimetric analyzer  at a heating rate of 10 °C/min from 25 °C to 800 °C under a nitrogen flow. Differential thermogravimetry (DTG) curves were calculated from the TGA results. The tribological measurements were performed using a four-ball tester  under a load of 392 N at a rotating speed of 1450 r/min for 1 h at room temperature. Prior to testing, the steel bearing balls GCr15  were cleaned ultrasonically for 30 min with ethanol. The average values of coefficient of friction (COF) were reported from three measurements of each sample.−1 corresponded to the –OH stretching vibration and H–O–H bending vibration of adsorbed water within MMT interlayer, respectively. The characteristic absorption peak at 3632.5 cm−1 was attributed to the stretching vibration of the Al–OH and Si–OH in the silicate layers, and this –OH group was responsible for the reaction with the silane coupling agent [−1 were representative of the stretching vibration of Si–O and Si–O–Al, respectively. In addition of these characteristic peaks of MMT, novel vibration bands related to surfactants and/or silane coupling agents appeared in the spectra of OMMT  of pure PS was 383 °C. As for the PS matrix filled with OMMT, the onset decomposition temperatures of PS/P-MMT, PS/P-SLS-MMT, and PS/P-KH570-MMT were about 399 °C, 406 °C, and 416 °C, respectively, that was, 16 °C, 23 °C, and 33 °C higher than that of pure PS. The maximum mass loss temperature  was raised from 419 °C for the pure PS to 440 °C, 443 °C, and 449 °C for PS/P-MMT, PS/P-SLS-MMT, and PS/P-KH570-MMT, respectively. The enhancement in the thermal stability was due to the MMT layers forming a three-dimension network and a labyrinth pathway in the clay-polymer nanocomposite to prevent out-diffusion of the volatile decomposition products during thermal decomposition [T10% and Tmax of PS/P-SLS-MMT were higher than those of PS/P-MMT. The reason was perhaps that the maximum mass loss temperature of P-SLS-MMT was higher than that of P-MMT, as shown in T10% and Tmax of the PS/P-KH570-MMT were higher than those of the PS/P-SLS-MMT. This result indicated that the double bonds of silanes grafting on the edges and surfaces of the MMT could react with styrene monomer increased chemical interaction between the MMT and the polymers, resulting in the improved interface interactions and enhanced the thermal stability [tability . In thisTo further characterize the tribological properties for the PS/OMMT nanocomposites, we examined the wear surfaces of the PAO and PAO-PS/OMMT nanocomposites using SEM. Tmax increased from 419 °C of pure PS to 449 °C of PS/P-KH570-MMT. Moreover, the COF values of PAO-PS/OMMT nanocomposites were well below than that of pristine PAO, indicating increased friction reduction properties. Available results in this work can provide significant guidance for the future synthesis and application of the PS/OMMT nanocomposites in the oil and gas drilling engineering field to improve drilling fluid lubrication.Novel PS/OMMT nanocomposite particles were synthesized by soap-free emulsion polymerization in the presence of doubly organo-modified MMTs and investigated. The anionic surfactant, SLS, could be effectively used to further increase the interlayer space of P-MMT by the electrostatic interaction. The silane coupling agent, KH570, could be introduced to the external surfaces and broken edges of the P-MMT due to the silylation reaction. The complete exfoliation of the OMMT within the PS matrix was confirmed by XRD and TEM analysis. The introduction of OMMT significantly decreased the average particle size and PDI values of PS, as evidenced by DLS and SEM results, and this became more pronounced in the PS/OMMT nanocomposites filled with P-KH570-MMT. The results of TGA-DTG analysis confirmed that the thermal stability of the PS/OMMT nanocomposites was significantly enhanced compared with pure PS. The


Trypanosoma cruzi, the etiologic agent of Chagas disease, immunosuppression, and apoptosis of mature lymphocytes contribute to the establishment of the parasite in the host and thereby to persistence and pathology in the chronic stage of infection. In a systemic mouse model of experimental Chagas disease, we have demonstrated a strong depletion of mature B cells in the spleen during the first 2 weeks of infection. Remarkably, the decrease in this cell population commenced already in the bone marrow from infected mice and was a concomitant of an increased apoptosis in pro- and pre-B cell populations. Pro- and pre-B cells in the bone marrow showed a significant reduction accompanied by a functional disturbance of bone marrow-derived stromal cells resulting in diminished levels of IL-7, an essential factor for the development of B cell precursors. Ex vivo, stromal cells isolated from the bone marrow of infected mice had a strikingly impaired capacity to maintain the development of pro- and pre-B cells obtained from uninfected animals. Together, the reduction of an active humoral immune response during acute Chagas disease suggests to be an initial immune evasion mechanism of the parasite to establish persistent infection. Therefore, prevention of B cell depletion by rescuing the stromal cells during this early phase, could give rise to new therapeutic approaches.After infection with  Trypanosoma cruzi, the etiologic agent of Chagas disease . Acidified water (pH 3.0) and food were provided T. cruzi were kept cryopreserved , naïve sex- and age-matched mice were used.For 4Cl, 10 μM EDTA, 1 mM Na2CO3) and FCS was subsequently added . Fluorochrome-labeled streptavidin and the apoptosis marker merocyanine  were incubated separately. Samples were subsequently acquired on a FACSCalibur (BD Bioscience) and analyzed using the CellQuest software (BD Bioscience).Single cell suspensions were prepared and washed in ISCOVES medium. After centrifugation, erythrocytes were lysed in Red Cell Removal Buffer .Because of cell to cell action of secreted cytokines such as IL-7 or IL-3 in B cell development measurement on protein level was not suitable, therefore we focused on the gene expression level. For RNA extraction, bone marrow was isolated from mice at days 6, 10, 14, 21, and 32 after infection. Day 6 after infection was used as an infection-internal reference. After homogenization in solution D  suspensions were acidified in 2 M sodium acetate, purified by extractions with phenol/chloroform, precipitated with isopropanol, and washed twice in 70% ethanol. Air-dried RNA was dissolved in RNase-free H5 cpm/μl) in hybridization buffer and added (2 μl) to tubes containing extracted RNA from bone marrow of T. cruzi-infected mice dissolved in 8 μl of hybridization buffer. Samples were overlaid with mineral oil, heated to 90°C, and incubated at 56°C for 16 h. Single-stranded RNA was then digested by addition (100 μl) of RNase A (80 ng/μl) and RNase T1 (250 U/μl) in RNase buffer. After incubation for 45 min at 30°C, samples were treated for 15 min at 37°C with 18 μl of a mixture of proteinase K (0.3 mg/ml), yeast RNA (0.06 mg/ml), and proteinase K buffer to stop digestion. dsRNA was isolated and precipitated as above, dissolved in loading buffer, and electrophoreses was performed in standard 6% acrylamide/8 M urea sequencing gel. Dried gels were placed on BioMax film  with intensifying screens and were developed at −70°C for 72 h.Unlabeled sense RNA for IL-3, SCF, GM-CSF, IL-7, and L32 and all reagents were supplied by BD PharMingen (RiboQuant). For the synthesis of radiolabeled antisense RNA probes, the final reaction mixture (20 μl) contained 120 μCi of [α-32P]UTP ; UTP (61 pmol), GTP, ATP, and CTP (2.75 nmol each); 100 nmol DTT; transcription buffer (1 ×) RNasin (40 U); T7 RNA polymerase (20 U); and an equimolar pool of the template set. After 1 h at 37°C, the reaction was terminated by incubation with DNase (2 U) for 30 min at 37°C. Probes were purified by extractions with phenol/chloroform and precipitated with ethanol. Air-dried probes were dissolved . Resulting densities of the genes of interest were standardized against L32.2, 37°C). Non-adherent cells were removed and the adherent cells were incubated for additional 3 days. Cultures of stromal cells were checked microscopically and images were taken by a computer aided camera.For the examination of stromal cells, we isolated the whole bone marrow of both femurs from uninfected and infected mice by flushing with a 1 ml syringe into ice cold ISCOVES medium. For any given day post infection bone marrow obtained from uninfected mice were used as control. Erythrocytes were eliminated with RCRB and the remaining bone marrow cells were washed and cultivated for 3 days , L-arginine (Sigma Aldrich), the arginase inhibitor N-hydroxy-L-arginine (NhLA) (Sigma Aldrich), and IL-4 . Subsequently, a cellular stress assay was performed  following the manufacturer's instructions .t-test in Graphpad Prism .Quantifiable data are expressed as the means of individual determinations and standard deviations. Statistical analysis was performed using, dependent on the data analyzed, Wilcoxon signed rank test, Mann–Whitney U test, or the two-tailed Student's T. cruzi trypomastigotes flow-cytometric analysis revealed an unchanged frequency of 40% B cells in the spleen of C57BL/6 mice  was reduced in the further course of infection reaching the lowest frequency 14 days post infection , the proportion of apoptotic B cells significantly increased after infection with a maximum at day 14 post infection  and c-kit as well as via cytokines released from stromal cells , 33. Pars Figure . Whereass Figure  was dense Figure  inset, De Figure . BecauseT. cruzi after ex vivo cultivation of bone marrow stromal cells for 72 h. In microscopy, the stromal cell population of the bone marrow was able to reach confluency until 7 days post infection but collapsed thereafter  or 10 days similarly promoted B cell development as measured by the frequencies of pro- and pre B cells  and arginase-1 by the cytokines IFN-γ and IL-4, respectively (T. cruzi-infected mice has an effect on stromal cells isolated from uninfected mice. In contrast to medium alone and serum of uninfected animals, serum derived from infected mice induced a strong stress response in stromal cell cultures (Figure It has been reported that L-arginine has a major impact on the differentiation of B cells . In thiselopment . Becauseectively –40 we wes Figure . SupplemT. cruzi, B cell activation results in the production of non-specific antibodies that fail to bind parasite antigens (T. cruzi infection. Hence, in Chagas disease a continuous polyclonal activation of lymphocytes ends up in anergy of these cells (T. cruzi-infected mice. Importantly, the amount of transitional B cells that come from the bone marrow was also reduced, thus explaining why the loss of B cells in the spleen was not replenished. Therefore, we examined the development of B cells in the bone marrow by flow cytometry, and also the numbers of pro-, pre-, and immature B cells were significantly diminished. Hence, the increased apoptosis of B lymphocytes in the spleen (In the acute phase of an infection with antigens . Moreoveantigens , 43 and antigens  and -indantigens , illustrse cells . In addise cells , 46, 47.e spleen  appears e spleen , 50, desT. cruzi (T. cruzi-infected mice (T. cruzi affects this function of bone marrow stromal cells. Concomitant with the impaired development of B cell precursors and immature B cells, we show that the integrity of the bone marrow stroma was affected during acute T. cruzi infection. B cell precursors, such as pro- and pre-B cells, interact with stromal cells via receptor-ligand complexes and cytokines (Il3, Gm-csf, Scf, and Il7 gene expression in the bone marrow of T. cruzi-infected mice in our study further points at a dysfunction of stromal cells impacting on B cell development. B cell development depends also as aforementioned on the direct interaction of precursors with stromal cells via receptors and ligands (T. cruzi-infected mice lost the capacity to promote B cell differentiation. Because pre-B cells can differentiate into immature B cells in vitro only in the absence of IL-7 and stromal cells (T. cruzi induces a dysfunction of bone marrow stromal cells which is responsible for transient diminished B cell development during the acute infection. This alteration in the development of B cells contributed to the overall loss of this lymphocyte population in the periphery.Besides polyclonal B cell activation, also bone marrow hypoplasia has been described in bacterial , 52, funT. cruzi . In geneted mice  suggestited mice , 58–64 wytokines , 58, 65.ytokines , 66, 67.ytokines . Hence,  ligands , 62. To al cells  our resuT. cruzi-infected mice that may contribute to stromal cell alterations during the acute phase of Chagas disease (Figure T. cruzi infection. Elderly mice that are more susceptible to a T. cruzi infection express elevated levels of arginase in contrast to the more resistant younger individuals (T. cruzi antigen, cruzipain (T. cruzi infection (In addition to this direct infection-induced effect, the hyperinflammation during acute Chagas disease may also lead to reduced viability of stromal cells by apoptosis as is described for cells facing nutritional deprivation . A strone Figure . In thise Figure . Becausee Figure  the IL-4ividuals . In addiruzipain  is able ruzipain . In thisnfection . In the nfection  and the T. cruzi with the immune system that presumably foster a timely immune evasion of the parasite during the acute phase of infection by reducing the number of antibody-producing cells in the periphery through a developmental diminishment of their precursors already in the bone marrow. The transient changes in stromal cell function and subsequent loss of B cell precursors may appear to contribute to establish the parasite in the host. However, restoring the stromal cell integrity during the acute phase of T. cruzi infection would experimentally proof that the inflammation-induced alteration of stromal cell function in the bone marrow really affects B cell development and subsequently effector responses against the parasite. Together, rescuing the B cells and its precursors during the acute infection may limit the risk of a life-long infection by T. cruzi and the subsequent sequelae in the chronic phase of Chagas disease.Together, our data highlight the complex interplay of UM performed the experiments, analyzed the data, and wrote the manuscript. GS conceived the project. HM conceived the project. GK analyzed the data. RC conceived and designed the experiments and wrote the manuscript. CH conceived and designed the experiments and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Long poly-diacetylene chains are excellent candidates for planar, on-surface synthesized molecular electronic wires. Since hexagonal-Boron Nitride (h-BN) was identified as the best available atomically flat insulator for the deposition of poly-diacetylene precursors, we demonstrate the polymerization patterns and rate on it under UV-light irradiation, with subsequent polymer identification by atomic force microscopy. The results on h-BN indicate self-sensitization which yields blocks comprised of several polymers, unlike on the well-studied graphite/diacetylene system, where the polymers are always isolated. In addition, the photo-polymerization proceeds at least 170 times faster on h-BN, where it also results in longer polymers. Both effects are explained by the h-BN bandgap, which is larger than the diacetylene electronic excitation energy, thus allowing the transfer of excess energy absorbed by polymerized wires to adjacent monomers, triggering their polymerization. This work sets the stage for conductance measurements of single molecular poly-diacetylene wires on h-BN. In search of materials for the electronics of the future, conjugated polymers ,2,3 in gn ,15,16,17n ,18. Thisn  drug deln .Polymerized diacetylene chains are therefore excellent candidates for the on-surface fabrication of planar molecular electronic circuits. For this reason, the self-assembly of the PDA precursors must occur: (1) in a flat-lying manner to ensure device planarity; and (2) on an atomically flat insulator as the molecules are sensitive to surface corrugation  and for It was recently shown ,23 that 2 chips. After exfoliation we annealed the substrates at 500 °C for 2 h for adhesive tape residue and ambient adsorbate removal . 3(CH2)15C≡C–C≡C(CH2)8COOH), are amphiphilic with a hydrophilic COOH group at one end and a hydrophobic alkyl chain at the other end. The solution was prepared by adding 0.15 g/L of 10,12-nonacosadiynoic acid powder  in xylene. Note that this concentration has been optimized for the systematic formation of SAMs with minimal molecular debris accumulation elsewhere [After substrate preparation, surface-deposition of the diacetylene molecules was performed by dropcasting a 4 μL droplet on the sample followed by spin-coating at 8000 rpm for 90 seconds. The diacetylene molecules used, 10,12-nonacosadiynoic acid . Experiments were performed using a Keysight 5500 microscope  and Multi75-G silicon cantilevers  with 3 nm2). Exposure time varied from few seconds (h-BN) to several minutes (HOPG). Details on the UV-polymerization process of diacetylene can be seen in the literature [Photo-polymerization was performed by irradiating the samples under a low-pressure mercury UV lamp 11C≡C–C≡C(CH2)8COOH) on HOPG after 20 min [terature , the strterature ,28. Lengr 20 min  irradiat2) and corresponding phase shift, respectively, for diacetylene on h-BN after 5 s of irradiation. The stripe period is identical to the HOPG case. Several PDA chains with lengths under 100 nm can be identified. In addition, there is one polymer 160 nm long. Similar to the HOPG surface, the polymers appear higher than the surroundings. Polymerization rate on h-BN seems significantly faster as compared to HOPG. To confirm this point, we irradiated the same sample for five more seconds . AFM topography and phase shift are presented in −1 [−1 are attributed to the –C=C– stretching modes of the polymer backbone [−1 are due to –C≡C– backbone stretching variations [−1 [−1 . In the backbone ,34,35. Sriations ,37. In Fions [−1 . Note thAfter the second irradiation, PDA chains on h-BN appear next to each other, effectively forming islands of polymerized material. This is attributed to the well-known effect of self-sensitization, which causes partially polymerized crystals to further polymerize slightly faster than pure monomer crystals. The mechanisms proposed to date include reactivation of the chain end , homogenWp in mW in Here, we propose a mechanism as shown in In 2, as its bandgap (1.2 eV) is smaller than the diacetylene excited state energy which also allows it to relax results in a similar photo-polymerization rate as the HOPG system. However, the large bandgap of h-BN suppresses the relaxation mechanism, leading to more on-surface reactions that are translated to more polymers on the surface of h-BN. Polymer count as a function of time, on both substrates, and averaged from several experiments, is summarized in The reason for the enhanced photo-polymerization rate of diacetylene on h-BN vs. HOPG is also attributed to the large electronic bandgap of the former. Since the excitation energy of diacetylene is about 3 eV, it can relax on the HOPG substrate as graphite has no bandgap. This is corroborated by photo-polymerization on MoSBesides the polymer count after irradiation, we also observed a significant difference in the averaged polymer length between substrates. On HOPG, the averaged polymer length is slightly increasing or stays almost constant within the error: 28 ± 7 nm for 10 min and 42 ± 16 nm for 20 min irradiation, corresponding to 60 and 90 monomers per polymer chain, respectively  is necessary for efficient layer polymerization. As a next step, the stage is ready for the fabrication of simple source-drain devices based on h-BN and PDA for conductance measurements, initially via self-sensitized polymer islands, and finally through a single PDA chain.


Co-infection of hepatitis B virus (HBV) and hepatitis C virus (HCV) with human immunodeficiency virus (HIV) is associated with increased risk of hepatic complications and mortality. A retrospective study to estimate the proportion of HBV and HCV co-infections in Singapore was conducted using a clinical database. We included 3065 patients who were seen under the Clinical HIV Programme at the largest referral centre for HIV care between 2006 and 2017 and were tested for both HBV and HCV. Factors associated with HIV-HBV and HIV-HCV co-infections were determined using logistic regressions. The majority (86.3%) of HIV-infected patients were mono-infected, while 7.2% were co-infected with HBV, 6.0% with HCV, and 0.5% were co-infected with both HBV and HCV. The most common HCV genotype was GT1 (63%). Factors significantly associated with HBV co-infection in the multivariable model were: Aged 30–49 years and 50–69 years at HIV diagnosis, male gender, and HIV transmission through intravenous drug use (IDU). Independent factors associated with HCV co-infection were: Malay ethnicity, HIV transmission through IDU, and HIV diagnosis between 2006 and 2008. Behavioural risk factors such as IDU, as well as epidemiologic differences associated with co-infection, should inform further studies and interventions aimed at reducing viral hepatitis infection among HIV-infected individuals.  Human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) are the most common chronic viral infections worldwide . These tPrior to the availability of highly active antiretroviral therapy (HAART), patients with HIV-HBV co-infection were 19 times more likely than patients mono-infected with HBV to die from liver-related complications . The advApproximately 37 million people worldwide are infected with HIV, and about 7.4% of them are co-infected with HBV . The estThe estimated prevalence of HCV infection worldwide is 2.2% , while tIn Singapore, about 450 new cases of HIV are reported every year, and this number has remained fairly constant since 2008, with notification rates of 103.7 to 125.2 per million resident population . The majA local study investigating the risk factors and time trends for co-infections in a random sample of over 700 HIV-infected patients who were referred for first-time care from 2006 to 2011 found that 8.1% were co-infected with HBV and 2% had active HCV co-infection . It is iThe Clinical HIV Programme at the National Centre for Infectious Diseases (NCID) sees the highest number of HIV-infected patients in Singapore. The NCID is a 330-bed purpose-built facility and houses clinical services, public health, research, training, and education under one overarching structure. We made use of a clinical database maintained by the programme for the retrospective study, with the following information being routinely collected for all patients referred for care: Demographics, virologic and immunologic parameters, antiretroviral therapy (ART) and monitoring, and routine biochemical test results performed in the course of HIV care. The study population comprised Singapore residents who were diagnosed with HIV and attended the NCID at least once between 2006 and 2017. We confined the study subjects to HIV-infected patients who were tested for both HBV and HCV. HIV infection was confirmed by western blot assay following a positive result from a fourth-generation chemiluminescent microparticle immunoassay (CMIA). HBV infection was ascertained based on seropositivity for the hepatitis B antigen surface (HBsAg) using electrochemiluminescence immunoassay (ECLIA), and hepatitis B e-antigen (HBeAg) was also measured using ECLIA. HCV infection was based on seropositivity for the anti-HCV antibody (anti-HCV) test using ECLIA, or detectable HCV with real-time polymerase chain reaction (PCR). HCV genotyping was done by reverse line probe hybridisation.Co-infection means an affected person is living with two or more infections at the same time. Individuals should not be considered as having both HIV and HBV and/or HCV infections simultaneously at any time point if they have been cleared of HBV or HCV prior to their HIV diagnosis. Approximately 95% of adults newly infected with HBV recover completely and do not become chronically infected ,17. AfteEpidemiological characteristics and laboratory parameters were compared in four groups of HIV-infected patients: HIV mono-infection, co-infections with HBV only, co-infections with HCV only, and HIV-HBV-HCV co-infections.We calculated the 95% confidence intervals (CIs) for binomial proportions using Wilson’s method. The Chi-square test was used to test for group differences. Proportions between two groups were compared using two-sample independent z tests, with standard error estimated using pooled value of the two proportions. The Mantel-Haenszel linear-by-linear association Chi-square test was used to evaluate whether there was linear trend in proportion of co-infections over time.p > 0.10 for removal of variables. All variables with p < 0.05 were retained in the final multivariable model.The main outcomes of interest were whether the patient had HBV or HCV co-infections or not. For univariable logistic regression analyses, odds ratios (OR) and 95% CIs were calculated. Multivariable logistic regression was used to determine independent risk factors associated with co-infections, and adjusted odds ratios (aOR) were calculated. Variables were considered for inclusion in the multivariable model using the likelihood-ratio statistic based on the maximum partial likelihood estimates in backward stepwise selection with The Kaplan-Meier method was used to estimate the five-year survival rate. All reports of deaths, irrespective of cause, which occurred by the end of 2017 were included in the survival analysis. Patients contributed person-time from the date of HIV diagnosis until death or 31 December 2017, whichever was the earlier date. p values reported were two-sided, and statistical significance was taken as p < 0.05. Statistical analyses were performed using the IBM SPSS Statistics for Windows, version 24 .All Ethical approval for use of the clinical database was obtained from the Singapore National Healthcare Group Domain Specific Review Board (NHG DSRB reference number 2012/00438). Informed consent was not obtained as the clinical data collected was used as part of the care management of HIV patients. All data analysed for the study were anonymised. A total of 3327 HIV-infected patients were seen under the Clinical HIV Programme from 2006 to 2017. There were 3139 patients who had been tested for HBV, and among them, 3097 had also been tested for HCV. After excluding 32 who had been tested positive for HBV and/or HCV more than one year before HIV diagnosis and subsequently tested negative, a total of 3065 HIV-infected patients constituted the final sample for this study. The proportion of HBV co-infection among HIV-infected patients was 7.7% (95% CI: 6.8–8.7%), while the proportion with HCV co-infection was 6.6% (95% CI: 5.7–7.5%). The majority (86.3%) were mono-infected, while 0.5% were co-infected with HBV and HCV. The mean age at HIV diagnosis was 40.2 years . Adults aged 20–49 years at HIV diagnosis comprised about 74.3% of the study sample. The majority (93.8%) were men, and over three-quarters (76.0%) were Chinese. The main mode of HIV transmission was via sexual exposure (96.6%). Most of the HIV-infected patients (95.5%) were on antiretroviral treatment at the time of analysis.Patients with triple infections of HIV, HBV, and HCV were diagnosed with HIV at a younger age ; the majority (87.6%) were aged 20–49 years at diagnosis . About tp = 0.01) (p = 0.003) and the group who were co-infected with HCV only (63.2%) (p < 0.005). Patients of Malay ethnicity comprised 30.3% of those co-infected with HCV only, and this proportion was significantly higher than that in the HIV mono-infected group (15.1%) and in the group who were co-infected with HBV only (10.0%) (both p < 0.0005).In the HIV mono-infected group, 93.3% were men—a significantly lower proportion compared to 97.7% in those co-infected with HBV only ( = 0.01) . Patientp < 0.0005).Across the four groups, the majority were infected via sexual mode of transmission . Among pp = 0.01) (A significantly higher proportion of patients co-infected with HCV (40.0%) were diagnosed with HIV in the period 2006–2008, compared to 28.3% in the mono-infected group ( = 0.01) . In the p < 0.0005). About one-quarter of patients co-infected with HCV only (24.9%) tested positive for HCV more than 48 months after HIV diagnosis. In the group with triple infections, about 37.5% had HIV and either HBV and/or HCV diagnosed within one month. Among patients co-infected with HBV only, about 57.3% had both infections diagnosed within one month . Among pp = 0.037) (p = 0.003).There was a significant decline in the proportion of HIV-infected patients who were co-infected with HBV over the period of HIV diagnosis; from 8.8% in 2006–2008 (95% CI: 7.1–10.8%) to 5.5% in 2015–2017 (95% CI: 4.0–7.7%)  . The pro3, which was significantly higher compared to 46.1% in the HIV mono-infected group (p = 0.005) and 39.8% in those co-infected with HCV only (p = 0.002) (p < 0.0005). Among patients with alanine aminotransferase (ALT) measured at HIV diagnosis, about 21.4% of those co-infected with HBV only had ALT > 55 U/liter, which was significantly higher compared to 10.5% in the HIV mono-infected group (p < 0.0005).Among patients with CD4 measured at HIV diagnosis, over half (56.3%) of those co-infected with HBV only had CD4 count ≤ 200cells/mm= 0.002) . Among pOf the 236 HIV patients co-infected with HBV, 216 (91.5%) underwent tenofovir-containing ART. Among the 180 patients tested positive for HBsAg who were also tested for HBeAg during the corresponding period, 85  were positive for HBeAg.Univariable logistic regression analyses indicated that relative to HIV mono-infected patients, the factors associated with the presence of HBV co-infection were: Being in the age groups of 30–49 and 50–69 years, male gender, Chinese ethnicity, IDU as the mode of HIV transmission, diagnosis of HIV being made in the period 2006–2008, being diagnosed with AIDS-defining illnesses/opportunistic infections within one year of HIV diagnosis, and history of using recreational or illicit drugs .Multivariable logistic regression identified age group, gender, and mode of HIV transmission as significant factors that were independently associated with HBV co-infections among HIV-infected patients. Compared to patients diagnosed at 10–29 years of age, those who were diagnosed with HIV at 30–49 years  and 50–69 years  were at higher odds of HBV co-infections. The odds of HBV co-infections were significantly higher among men than women . Compared to patients infected via sexual mode of transmission, those who were infected via IDU had over four times the odds of HBV co-infections .Factors associated with the presence of HCV co-infections in univariable logistic regression analyses were: Malay ethnicity, IDU as the mode of HIV transmission, diagnosis of HIV in the period 2006–2008, and history of using recreational or illicit drugs . In multOf the 201 HIV-infected patients co-infected with HCV within this study, 129 of them had HCV genotype testing. Excluding six with indeterminate genotype results, the common HCV genotype (GT) was GT-1 (63%), followed by GT-3 (32%) among 123 HIV-infected patients. p = 0.06).The cumulative proportion of patients in the HIV mono-infected group surviving until the fifth year since HIV diagnosis was 93%, while it was 89% in those co-infected with HBV only and 91% in those co-infected with HCV only. There was no statistical difference in survival time between these three groups   and GermThe proportion of HCV co-infection among HIV-infected patients in this study was 6.0%. In comparison, the overall prevalence of HIV-HCV co-infection in Western Europe and the United States was 25% to 30%  and 15.2The significant risk factors associated with HIV-HBV co-infection in this study were being aged 30-49 years and 50-69 years at HIV diagnosis, male gender, and HIV transmission via IDU alone . In SoutPatients with HIV-HBV co-infection were diagnosed at an older age compared to those with HIV-HCV co-infections. There was a significant decline in the proportion of patients who were co-infected with HBV over the period of HIV diagnosis, from 8.8% in 2006–2008 to 4.5% in 2015–2017. These two findings could be attributed to the introduction of hepatitis B vaccination as part of the NCIP; hepatitis B vaccination was introduced into the programme for babies born to hepatitis B-carrier mothers on 1 October 1985, and subsequently extended to all newborns on 1 September 1987 . HIV transmission via IDU was also a significant risk factor independently associated with HIV-HCV co-infection in our study . Our stu® (or buprenorphine hydrochloride) was approved by MOH as a substitute treatment for opiate-dependent drug abusers [Besides IDU as the mode of HIV transmission, Malay ethnicity and being diagnosed with HIV between 2006 and 2008 were also associated with HIV-HCV co-infections . These r abusers . However abusers . Subsequ abusers . As of t abusers . The epi abusers .Patients with HIV-HCV co-infection were diagnosed at a younger age compared to HIV-HBV co-infection. The younger age seen in patients infected via IDU could be explained by the pattern of drug use in Singapore: About 64% of drug abusers arrested in 2017 were below the age of 30 years [The most common HCV genotype seen in our study was GT-1, followed by GT-3. In a study on residual blood serum samples of blood donors screened between 2011 and 2014, 0.06% were positive for HCV, and of the 42 serum samples available for genotyping, the distribution was 48% GT-3 and 31% GT-1 . RegionaThere are limitations to this study. Our study is confined to patients followed-up at the NCID. However, as the NCID is the largest referral centre for HIV care, our study population could be considered as fairly representative of newly diagnosed HIV patients at large. Tests for HBV and HCV are mostly carried out on order from the treating physician, hence patients who did not have these two tests had to be excluded from the study. Treatment history was not available in the clinical database and we were unable to determine whether the HBV and/or HCV infection was chronic or acute. Patients with chronic HBV and/or HCV infection who had been tested positive more than one year prior to their HIV diagnosis could have been mis-classified as having acute infection and they were excluded from our study, but this number was expected to be small in view of the considerable proportion who would have achieved spontaneous viral clearance within six months of infection, particularly for HBV ,17,18,19The proportion of viral hepatitis co-infections in patients living with HIV could be higher, but as Singapore follows the United States Department of Health and Human Services (DHHS) guidelines for management of HIV, almost all patients are screened for co-infections upon HIV diagnosis . As partGiven that IDU is the only modifiable risk factor for HIV-HCV co-infection in our study, it is important to consider interventions to control the spread of infections via this route. One of the key interventions that has been implemented in the United States and Europe to reduce the risk of transmission of HIV and other blood-borne viruses among PWID is harm reduction programmes, such as needle exchange programmes. Participation in these programmes was associated with reduction in the risk of HIV incidence by 33% in the United States, and by two to three folds in Amsterdam ,36. The In conclusion, it is encouraging that there is a declining trend of co-infection with both HBV and HCV across the years, especially given that the number of new HIV diagnoses remains consistent since 2008. Many of the patients co-infected with HIV and HCV or triple infected were diagnosed with HIV at a younger age, reflecting the higher proportion of PWID seen in younger age groups. While the number of PWID in Singapore is low, it is still important to address the issue of IDU: Compared to patients infected with HIV via the sexual route, those who were infected via IDU had over four times the odds of HBV co-infections and 20 times the odds of HCV co-infections. In the absence of a HCV vaccine, other preventive interventions—such as risk-stratified screening, testing and treating, and behavioural interventions—are needed as part of hepatitis C control efforts in Singapore .